Identification of HLA-E Binding Mycobacterium tuberculosis-Derived Epitopes through Improved Prediction Models.

Tuberculosis (TB) remains one of the deadliest infectious diseases worldwide, posing great social and economic burden to affected countries. Novel vaccine approaches are needed to increase protective immunity against the causative agent Mycobacterium tuberculosis (Mtb) and to reduce the development of active TB disease in latently infected individuals. Donor-unrestricted T cell responses represent such novel potential vaccine targets. HLA-E-restricted T cell responses have been shown to play an important role in protection against TB and other infections, and recent studies have demonstrated that these cells can be primed in vitro. However, the identification of novel pathogen-derived HLA-E binding peptides presented by infected target cells has been limited by the lack of accurate prediction algorithms for HLA-E binding. In this study, we developed an improved HLA-E binding peptide prediction algorithm and implemented it to identify (to our knowledge) novel Mtb-derived peptides with capacity to induce CD8+ T cell activation and that were recognized by specific HLA-E-restricted T cells in Mycobacterium-exposed humans. Altogether, we present a novel algorithm for the identification of pathogen- or self-derived HLA-E-presented peptides.

Thermal-exchange HLA-E multimers reveal specificity in HLA-E and NKG2A/CD94 complex interactions.

There is growing interest in HLA-E-restricted T-cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide-specific T cells has allowed the in-depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T-cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA-E monomers and multimers allowing the high-throughput production of HLA-E multimers. We optimized the thermal-mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high-throughput identification and analysis of HLA-E-binding peptides which could be involved in T-cell and NK cell-mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA-E with this receptor. In particular, our results reveal that interactions of HLA-E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA-E multimer technology based on thermal-mediated peptide exchange allowing us to investigate the molecular requirements for HLA-E/peptide interaction with its receptors.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

Peptide Binding to HLA-E Molecules in Humans, Nonhuman Primates, and Mice Reveals Unique Binding Peptides but Remarkably Conserved Anchor Residues.

Ag presentation via the nonclassical MHC class Ib molecule HLA-E, with nearly complete identity between the two alleles expressed in humans, HLA-E*01:01 and HLA-E*01:03, can lead to the activation of unconventional T cells in humans. Despite this virtual genetic monomorphism, differences in peptide repertoires binding to the two allelic variants have been reported. To further dissect and compare peptide binding to HLA-E*01:01 and HLA-E*01:03, we used an UV-mediated peptide exchange binding assay and an HPLC-based competition binding assay. In addition, we investigated binding of these same peptides to Mamu-E, the nonhuman primate homologue of human HLA-E, and to the HLA-E-like molecule Qa-1b in mice. We next exploited the differences and homologies in the peptide binding pockets of these four molecules to identify allele specific as well as common features of peptide binding motifs across species. Our results reveal differences in peptide binding preferences and intensities for each human HLA-E variant compared with Mamu-E and Qa-1b Using extended peptide libraries, we identified and refined the peptide binding motifs for each of the four molecules and found that they share main anchor positions, evidenced by conserved amino acid preferences across the four HLA-E molecules studied. In addition, we also identified differences in peptide binding motifs, which could explain the observed variations in peptide binding preferences and affinities for each of the four HLA-E-like molecules. Our results could help with guiding the selection of candidate pathogen-derived peptides with the capacity to target HLA-E-restricted T cells that could be mobilized in vaccination and immunotherapeutic strategies.

Interleukin-6-mediated resistance to immunotherapy is linked to impaired myeloid cell function.

High serum levels of interleukin-6 (IL-6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)-associated tumor model expressing IL-6 to investigate the impact of tumor-expressed IL-6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor-produced IL-6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor-produced IL-6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T-cell-mediated killing. Furthermore, no overt differences in the frequencies of tumor-infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL-6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL-6-mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL-6-mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer.

Detailed characterization of human Mycobacterium tuberculosis specific HLA-E restricted CD8+ T cells.

HLA-E presented antigens are interesting targets for vaccination given HLA-Es' essentially monomorphic nature. We have shown previously that Mycobacterium tuberculosis (Mtb) peptides are presented by HLA-E to CD8+ effector T cells, but the precise phenotype and functional capacity of these cells remains poorly characterized. We have developed and utilized in this study a new protocol combining HLA-E tetramer with intracellular staining for cytokines, transcription factors and cytotoxic molecules to characterize these cells in depth. We confirm in this study the significantly increased ex vivo frequency of Mtb-peptide/HLA-E-TM+ CD8+ T cells in the circulation of patients with active tuberculosis (TB). HLA-E restricted CD8+ T cells from TB patients produced more IL-13 than cells from controls or subjects with latent tuberculosis infection (LTBI). Compared to total CD8+ T cells, HLA-E restricted cells produced more IFNγ, IL-4, IL-10, and granulysin but less granzyme-A. Moreover, compared to "classical" Mtb specific HLA-A2 restricted CD8+ T cells, HLA-E restricted CD8+ T cells produced less TNFα and perforin, but more IL-4. In conclusion, HLA-E restricted- Mtb specific cells can produce Th2 cytokines directly.

The Breadth of Synthetic Long Peptide Vaccine-Induced CD8+ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection.

There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.

Potential of DosR and Rpf antigens from Mycobacterium tuberculosis to discriminate between latent and active tuberculosis in a tuberculosis endemic population of Medellin Colombia.

BACKGROUND: Tuberculosis (TB) remains one of the most deadly infectious diseases. One-third to one-fourth of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb) without showing clinical symptoms, a condition called latent TB infection (LTBI). Diagnosis of Mtb infection is based on the immune response to a mixture of mycobacterial antigens (PPD) or to Mtb specific ESAT-6/CFP10 antigens (IGRA), highly expressed during the initial phase of infection. However, the immune response to PPD and IGRA antigens has a low power to discriminate between LTBI and PTB. The T-cell response to a group of so-called latency (DosR-regulon-encoded) and Resuscitation Promoting (Rpf) antigens of Mtb has been proved to be significantly higher in LTBI compared to active TB across many populations, suggesting their potential use as biomarkers to differentiate latent from active TB. METHODS: PBMCs from a group LTBI (n = 20) and pulmonary TB patients (PTB, n = 21) from an endemic community for TB of the city of Medellín, Colombia, were in vitro stimulated for 7 days with DosR- (Rv1737c, Rv2029c, and Rv2628), Rpf- (Rv0867c and Rv2389c), the recombinant fusion protein ESAT-6-CFP10 (E6-C10)-, or PPD-antigen. The induced IFNγ levels detectable in the supernatants of the antigen-stimulated cells were then used to calculate specificity and sensitivity in discriminating LTBI from PTB, using different statistical approaches. RESULTS: IFNγ production in response to DosR and Rpf antigens was significantly higher in LTBI compared to PTB. ROC curve analyses of IFNγ production allowed differentiation of LTBI from PTB with areas under the curve higher than 0.70. Furthermore, Multiple Correspondence Analysis (MCA) revealed that LTBI is associated with higher levels of IFNγ in response to the different antigens compared to PTB. Analysis based on decision trees showed that the IFNγ levels produced in response to Rv2029c was the leading variable that best-classified disease status. Finally, logistic regression analysis predicted that IFNγ produced by PBMCs in response to E6-C10, Rv2029c, Rv0867c (RpfA) and Rv2389c (RpfA) antigens correlates best with the probability of being latently infected. CONCLUSIONS: The Mtb antigens E6-C10, Rv2029c (PfkB), Rv0867c (RpfA) and Rv2389c (RpfA), may be potential candidates to discriminate LTBI from PTB.

The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities.

Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at +1 and Tyr at +3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from -4 to +4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.

Local and systemic XAGE-1b-specific immunity in patients with lung adenocarcinoma.

XAGE-1b is a cancer/testis antigen aberrantly expressed in pulmonary adenocarcinoma. Systemic antibody and T cell responses have been demonstrated in adenocarcinoma patients, but so far, local antigen-specific immunity has not been reported. In this study, XAGE-1b expression by tumor cells as well as the presence of systemic and/or local XAGE-1b-specific immunity was assessed in peripheral blood, tumor tissue and tumor-draining lymph nodes of Caucasian patients with pulmonary adenocarcinoma. XAGE-1b protein expression was detected in 43.6% (17 of 39) of patients when at least two different parts of a resected tumor were assessed. In 20 patients, analysis of T cells isolated and expanded from the primary tumor and its draining lymph node demonstrated XAGE-1b-specific responses in two patients. XAGE-1b-specific immunoglobulin G antibodies were found in 3 of 40 patients. These three antibody-positive patients had also mounted a systemic T cell response to XAGE-1b, measured by proliferation, cytokine production and expression of T cell activation markers on peripheral blood mononuclear cells. The population of XAGE-1b-specific T cells comprised both CD4+ and CD8+ T cells secreting both type I and II cytokines. Epitope mapping showed that T cells predominantly targeted the N-terminal part of the XAGE-1b protein, while the B cell response was directed against the C-terminal domain. Our study for the first time provides evidence for the presence of XAGE-1b-specific T cells within adenocarcinoma tissue, which supports the concept that XAGE-1b acts as a genuine tumor antigen and, therefore, might form an attractive target for a vaccine-based approach of immunotherapy.

Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal.

BACKGROUND: Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. METHODS: To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. RESULTS: At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. CONCLUSIONS: This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.

An unbiased genome-wide Mycobacterium tuberculosis gene expression approach to discover antigens targeted by human T cells expressed during pulmonary infection.

Mycobacterium tuberculosis is responsible for almost 2 million deaths annually. Mycobacterium bovis bacillus Calmette-Guérin, the only vaccine available against tuberculosis (TB), induces highly variable protection against TB, and better TB vaccines are urgently needed. A prerequisite for candidate vaccine Ags is that they are immunogenic and expressed by M. tuberculosis during infection of the primary target organ, that is, the lungs of susceptible individuals. In search of new TB vaccine candidate Ags, we have used a genome-wide, unbiased Ag discovery approach to investigate the in vivo expression of 2170 M. tuberculosis genes during M. tuberculosis infection in the lungs of mice. Four genetically related but distinct mouse strains were studied, representing a spectrum of TB susceptibility controlled by the supersusceptibility to TB 1 locus. We used stringent selection approaches to select in vivo-expressed M. tuberculosis (IVE-TB) genes and analyzed their expression patterns in distinct disease phenotypes such as necrosis and granuloma formation. To study the vaccine potential of these proteins, we analyzed their immunogenicity. Several M. tuberculosis proteins were recognized by immune cells from tuberculin skin test-positive, ESAT6/CFP10-responsive individuals, indicating that these Ags are presented during natural M. tuberculosis infection. Furthermore, TB patients also showed responses toward IVE-TB Ags, albeit lower than tuberculin skin test-positive, ESAT6/CFP10-responsive individuals. Finally, IVE-TB Ags induced strong IFN-γ(+)/TNF-α(+) CD8(+) and TNF-α(+)/IL-2(+) CD154(+)/CD4(+) T cell responses in PBMC from long-term latently M. tuberculosis-infected individuals. In conclusion, these IVE-TB Ags are expressed during pulmonary infection in vivo, are immunogenic, induce strong T cell responses in long-term latently M. tuberculosis-infected individuals, and may therefore represent attractive Ags for new TB vaccines.

Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination.

Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low-dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)-related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen-specific migration of CD8+ T cells into HPV- and OVA-specific tumors. Local low-dose tumor irradiation alone resulted in a 2-fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10-fold and the number of CD8+ T cells specific for the E7- epitope increased more than 20-fold. Irradiation alone however also increased the number of intratumoral myeloid-derived suppressor cells (MDSCs) 3.5-fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen-specific CD8+ T cells and MDSCs in tumors increased up to 85-fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low-dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.

Longitudinal immune responses and gene expression profiles in type 1 leprosy reactions.

PURPOSE: Leprosy, a chronic disease initiated by Mycobacterium leprae, is often complicated by acute inflammatory reactions. Although such episodes occur in at least 50% of all leprosy patients and may cause irreversible nerve damage, no laboratory tests are available for early diagnosis or prediction of reactions. Since immune- and genetic host factors are critical in leprosy reactions, we hypothesize that identification of host-derived biomarkers correlated to leprosy reactions can provide the basis for new tests to facilitate timely diagnosis and treatment thereby helping to prevent tissue damage. METHODS: The longitudinal host response of a leprosy patient, who was affected by a type 1 reaction (T1R) after MDT-treatment, was studied in unprecedented detail, measuring cellular and humoral immunity and gene expression profiles to identify biomarkers specific for T1R. RESULTS: Cytokine analysis in response to M. leprae revealed increased production of IFN-γ, IP-10, CXCL9, IL-17A and VEGF at diagnosis of T1R compared to before T1R, whereas a simultaneous decrease in IL-10 and G-CSF was observed at T1R. Cytokines shifts coincided with a reduction in known regulatory CD39(+)CCL4(+) and CD25(high) T-cell subsets. Moreover, RNA expression profiles revealed that IFN-induced genes, (V)EGF, and genes associated with cytotoxic T-cell responses (GNLY, GZMA/B, PRF1) were upregulated during T1R, whereas expression of T-cell regulation-associated genes were decreased. CONCLUSIONS: These data show that increased inflammation, vasculoneogenesis and cytotoxicity, perturbed T-cell regulation as well as IFN-induced genes play an important role in T1R and provide potential T1R-specific host biomarkers.

Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation.

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome- and transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T-cell activation.

CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.

New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy.

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate-induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1ß, and IL-1ß in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1ß, and IL-1ß can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.

Excision Repair Cross-Complementation group 1 (ERCC1) C118T SNP does not affect cellular response to oxaliplatin.

AIMS: ERCC1 is involved in the repair of oxaliplatin-induced DNA damage. Studies for the association of the C118T SNP with clinical response to treatment with platinum drugs have rendered inconsistent results. We investigated the ERCC1 C118T SNP with respect to overall and progression-free survival in patients with advanced colorectal cancer (ACC) treated with oxaliplatin and in vitro DNA repair capacity after oxaliplatin exposure. In addition we discuss discrepancies from other studies concerning ERCC1 C118T. MATERIALS AND METHODS: Progression-free survival was determined in 145 ACC patients treated with oxaliplatin-based chemotherapy in a phase 3 trial. For the in vitro studies regarding ERCC1 functionality, we transfected an ERCC1 negative cell line with 118C or 118T ERCC1. Cellular sensitivity and DNA repair capacity after exposure to oxaliplatin was examined by Sulphorodamine B growth inhibition assay, COMET assay and Rad51 foci staining. RESULTS: We found no association between ERCC1 C118T and progression-free or overall survival. In addition, transfection of either 118C or 118T restores DNA-repair capacity of UV20 cells to the same level and chemosensitivity to oxaliplatin was similar in ERCC1 118C and 118T transfected cells. CONCLUSION: This study shows that the ERCC1 C118T variants are not associated with survival in ACC patients treated with oxaliplatin or the in vitro sensitivity and DNA-repair capacity in 118C and 118T transfected cell lines. Therefore, ERCC1 C118T genotyping seems of no value in individualizing oxaliplatin based chemotherapy in ACC.

Mtb HLA-E-tetramer-sorted CD8+ T cells have a diverse TCR repertoire.

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.

Inhibiting the NADase CD38 improves cytomegalovirus-specific CD8+ T cell functionality and metabolism.

Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in immunocompromised individuals such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in immunosuppressed individuals have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in immunocompromised patients and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with non-controlled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hypo-responsiveness in individuals who fail to control chronic viral infection.